Dialkoxybenzene and dialkoxyallylbenzene feeding and oviposition deterrents against the cabbage looper, trichoplusia ni: Potential insect behavior control agents

Article abstract of DOI:10.1021/jf9045123

The antifeedant, oviposition deterrent, and toxic effects of individual dialkoxybenzene compounds/sets and of hydroxy- or alkoxy-substituted allylbenzenes, obtained through Claisen rearrangement of substituted allyloxybenzenes, were assessed against the cabbage looper, Trichoplusia ni, in laboratory bioassays. Most of the compounds/sets strongly deterred larval feeding, with some exhibiting mild toxic and oviposition deterrent effects as well. Some of the compounds/sets were more active than the commercial insect repellent, DEET (N,N-diethyl-m-toluamide), as both feeding and oviposition deterrents against the cabbage looper. On the basis of the obtained oviposition data a general hypothesis was proposed regarding the oviposition sites: one binding mode with the alkyl and allyl groups on the same side of the benzene ring resulted in deterrence, the other with alkyl and allyl groups on opposite sides of the benzene ring resulted in stimulation. The results suggest some structure-activity relationships useful in improving the efficacy of the compounds and designing new, nontoxic insect control agents for agriculture.

Full text of DOI:10.1021/jf9045123

J. Agric. Food Chem. 2010, 58, 4983–4991 4983
DOI:10.1021/jf9045123
Dialkoxybenzene and Dialkoxyallylbenzene Feeding and
Oviposition Deterrents against the Cabbage Looper,
Trichoplusia ni: Potential Insect Behavior Control Agents
§
Y
ASMIN
A
KHTAR,† YANG
Y
U
,§ MURRAY B. ISMAN,*,† AND
E
RIKA
P
LETTNER
†Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia,
Canada V6T 1Z4, and §Department of Chemistry, Simon Fraser University, Burnaby, British Columbia,
Canada V5A 1S6
The antifeedant, oviposition deterrent, and toxic effects of individual dialkoxybenzene compounds/
sets and of hydroxy- or alkoxy-substituted allylbenzenes, obtained through Claisen rearrangement of
substituted allyloxybenzenes, were assessed against the cabbage looper, Trichoplusia ni, in
laboratory bioassays. Most of the compounds/sets strongly deterred larval feeding, with some
exhibiting mild toxic and oviposition deterrent effects as well. Some of the compounds/sets were
more active than the commercial insect repellent, DEET (N,N-diethyl-m-toluamide), as both feeding
and oviposition deterrents against the cabbage looper. On the basis of the obtained oviposition data
a general hypothesis was proposed regarding the oviposition sites: one binding mode with the alkyl
and allyl groups on the same side of the benzene ring resulted in deterrence, the other with alkyl
and allyl groups on opposite sides of the benzene ring resulted in stimulation. The results suggest
some structure-activity relationships useful in improving the efficacy of the compounds and
designing new, nontoxic insect control agents for agriculture.
KEYWORDS: Dialkoxybenzene; DEET; feeding deterrence; toxicity; oviposition sites; cabbage looper;
structure-activity; insect control agents
INTRODUCTION
can be applied to a host plant to prevent feeding or oviposition.
Therefore, deterrents have potential value in crop protection, in
combination with other strategies such as “attract and kill” (3).
Most natural plant defensive chemicals discourage insect
herbivory, either by deterring feeding and oviposition or by
impairing larval growth, rather than by killing insects outright.
Insect feeding deterrents can be found among all the major classes
of plant secondary metabolites: alkaloids, phenolics, and terpe-
noids (4). Especially well studied in this group are triterpenes such
as the limonoids from neem (Azadirachta indica), chinaberry
(Melia azedarach), and Citrus species and diterpenes, including
the clerodanes and the abietanes (5). Apart from terpenes,
another important class of compounds involved in the defense
ofplants against herbivoresand pathogens, as well as inattracting
pollinators, are the compounds often derived from aromatic
amino acids, phenolics (6).
Phenolic compounds can be short-range (polyphenols) or long-
range stimuli (phenylpropanoids) and are divided into hydrolyz-
able tannins (gallic acid esters of glucose and other sugars) and
phenylpropanoid-derived conjugates such as lignins, flavonoids,
and condensed tannins. The best studied polyphenols are the
flavonoids including flavonols, flavones, catechins, flavanones,
anthocyanidins, coumarins, and isoflavonoids (7, 8). Phenylala-
nine-derived phenolics include flavonoids, trans-cinnamic acid
and its derivatives, such as caffeic and ferulic acids, vanillin,
eugenol, and anethole (6, 9). Eugenol is a volatile member of the
phenylpropanoids from essential oils of many spices, particularly
For the past 40 years there has been an increased interest in the
behavioral manipulation of insect pests for their management, as
an alternative to broad-spectrum insecticides. Of particular interest
are nontoxic compounds that show some selectivity toward a pest
insect but not toward its natural enemies, pollinators, and the
environment. Successful manipulation of pest behavior could
provide protection of the resource (crop plant) through the use
of stimuli that either enhance or inhibit a particular behavior and
ultimately change its expression. The choice of a stimulus for
behavioral manipulation is usually dependent upon a number of
factors including accessibility, reproducibility, specificity, and
practicality (1).
Various short- or long-range stimuli, involved in behavioral
manipulation of insects, are perceived through contact chemore-
ceptors or olfactory receptors, respectively. These stimuli can either
stimulate feeding or oviposition, keeping the insect at the host plant,
or inhibit those behaviors, resulting in the insect abandoning the
plant. Examples of feeding stimulants include carbohydrates,
proteins, or fats (2) that are ubiquitous in plants, whereas oviposi-
tion stimulants can be highly species-specific. Feeding stimulants
can be used in conjunction with toxins in “attract and kill”
strategies (2), occasionally employed in crop protection. A deterrent
*Author to whom correspondence should be addressed [telephone
(604) 822-1219; fax (604) 822-6394; e-mail isman@interchange.
ubc.ca].
© 2010 American Chemical Society
Published on Web 03/12/2010
pubs.acs.org/JAFC

4984 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Akhtar et al.
Figure 1. (A) Synthesis of dialkoxybenzenes from catechol (1a), resorcinol (1b), or dihydroquinone (1c). Details of the synthesis and analyses have been
described previously (17). (B) Synthesis of Claisen rearranged products from 1-allyl-2-alkoxybenzenes. (C) Synthesis of Claisen rearranged products from
1-allyl-4-alkoxybenzene. (D) Synthesis of Claisen rearranged products from 1-allyl-3-alkoxybenzene.
clove (9). Cloves are useful in the home as moth deterrents
(personal observation), and the main odorant from cloves,
eugenol, is known to be perceived as a long-range stimulus by
several lepidopterans (10). One problem with phenylpropanoids
such as eugenol and compounds with a cinnamyl framework is
that they can produce toxic metabolites after benzylic/allylic
oxidation by certain cytochrome P450 enzymes (9).
Several polyphenolic compounds are also known for their
toxic/insecticidal effects (11). Flavonoids isolated from Annona
squamosa (12), Ricinus communis (13), and Calotropis procera (14)
are toxic to the pulse beetle, Callosobruchus chinensis, and
R. communis also caused oviposition deterrent and ovicidal effects in
addition to toxicity. Larvicidal activity of lignans, leptostachyol
acetate, and analogues from the roots of Phryma leptostachya
have been reported against three mosquito species (Culex pipiens
pallens, Aedes aegypti, and Ocheratatos togoi) (15).
The purpose of the present study was to assess the antifeedant,
oviposition deterrent, and toxic effects of candidate compounds
or compound sets including the individual candidate dialkoxy-
benzenes (Figure 1A) based on our previous structure-activity
work (16) indicating that compounds with intermediate group
sizes were the best lead compounds for oviposition and feeding
deterrent effects. Furthermore, the presence of a free hydroxyl
group as well as a methyl group served to reduce feeding deterrent
effects in all series of compounds, and replacement of a methyl
group with larger alkyl substituents increased thefeedingdeterrent
effects (in most cases) and substituted allylbenzene sets generated
by Claisen rearrangement of substituted allyloxybenzenes and
subsequent alkylation (Figure 1B-D) (17). The cabbage looper,
Trichoplusia ni, was chosen as a model insect for our study. The
cabbage looper is one of the most destructive insect pests of
vegetable crops. It feeds on a variety of crops including lettuce,

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 4985
beet, pea, celery, tomato, crucifers, certain ornamentals, and
weedy plants (18). Annual losses and the cost of control measures
make the cabbage looper an economically important pest (19).
Even though there are several natural enemies of cabbage looper,
chemical control is recommended in most situations (20). Because
T. ni has evolved resistance against many synthetic insecti-
cides (19) and the microbial insecticide Bacillus thuringiensis (21),
there is a need to develop new methods that could protect crops in
integrated pest management systems.
Here we have chosen to investigate relatively nontoxic com-
pounds that mimic naturally occurring bioactive odorants and
tastants and that are relatively easily prepared from commodity
chemicals. Because host plant detection is essential to the larval
and adult stages of this moth species (22), consequently leading to
crop damage, we target this chemical communication system with
aromatic odorants that interfere with larval feeding or the
oviposition behavior of adult moths, without causing acute toxic
effects to the insect.
90:10 hexane/toluene. To monitor the separation, 1% AgNO3 TLC plates
were prepared by running the silver nitrate solution up the plates and
drying them. The plates could be stained with anisaldehyde solution.
Isomer y ran more quickly than x, and it was possible to obtain several
fractions that contained pure y. However, y also tailed into the x peak, so
that it was difficult to obtain fractions with 100% x by FCC. Alternatively,
5b{3,1}y and 5b{3,1}x as well as 5b{3,2}y and 5b{3,2}x could be separated
by preparative TLC (100% hexanes) with multiple developments.
The more compact isomer y was more volatile than x, eluting usually
0.5-1 min earlier from the GC (DB-5 column). Also, in general, isomer y
formed an M þ 1 ion in the mass spectrum more readily and fragmented
more extensively (for example, to the tropylium ion m/z 91) than isomer x.
Data for the individual compounds and 1H NMR data of new, pure 3a,
3b, and 3c compounds that have not been published previously (17) are
given in the Supporting Information.
General Testing Procedure for the Bioassays. Initial screening for
feeding deterrent effects was conducted at 50 μg/cm2 in feeding deterrent
choice bioassays. Compounds exhibiting >50% feeding deterrence at this
concentration were subjected to further testing for oviposition deterrent
effects and contact toxicity at 0.25% of the test substance. For compounds
exhibiting >50% values for feeding deterrence, DC50 values (concent-
ration causing 50% feeding deterrence compared with the control) were
determined, on the basis of bioassays involving a minimum of four
concentrations (<3.12-25 μg/cm2).
MATERIALS AND METHODS
Plant Material. Cabbage plants (Brassica oleraceae var. Stonehead)
used in the bioassays were grown in plastic pots with a mixture of sandy
loam soil and peat moss (4:1) in a greenhouse at the University of British
Columbia (UBC), Vancouver, BC, Canada. Leaves used in the bioassays
were collected from 5-6-week-old cabbage plants.
Feeding Deterrent Bioassays. Leaf disk choice bioassays (16) were
conducted to determine feeding deterrent effects of the synthetic com-
pounds using freshly molted third-instar larvae starved for 4-5 h prior to
each bioassay. Larvae were given the choice of feeding on two leaf disks,
one treated with 10 μL of a solution of the test substance painted on each
side and the other treated with a carrier solvent alone. The number of
larvae was 25 per treatment. Bioassays were terminated when ∼50% of the
control disk had been eaten (normally 3-5 h).
Test Insects. T. ni larvae and moths were obtained from a long-
established colony (>50 generations) maintained on an artificial diet,
Velvetbean Caterpillar Diet (Bio-Serv Inc., Frenchtown, NJ) in the
insectary of UBC. It is therefore possible that our insects may respond
differently from wild conspecifics. The diet was supplemented with finely
ground alfalfa, to improve acceptability, and vitamins (Bio-Serv Inc.).
Test Compounds. Dialkoxybenzene compounds were synthesized
from catechol (1a), resorcinol (1b), or dihydroxyquinone (1c) as shown
in Figure 1. Synthesis of Claisen rearrangement products from 1-allyl-2-
alkoxybenzenes (B), 1-allyl-4-alkoxybenzenes (C), and 1-allyl-3-alkoxy-
benzenes (D) are shown in Figure 1 (17). Isomers x and y from the Claisen
rearrangement of meta-substituted allyloxybenzenes were separated by
flash chromatography on AgNO3-silica as described below.
Areas of control and treated leaf disks consumed by the larvae were
measured using Scion Image software, and feeding deterrence was
calculated (16) using the formula
½ðC - TÞ=ðC þ TÞꢀ ꢁ 100
where C and T are areas consumed of the control and treated leaf disks,
respectively.
Oviposition Deterrent Bioassays. Oviposition response of T. ni
moths was measured according to our previously described oviposition
choice bioassay (16). A pair of moths (one male and one female) was
introduced into an oviposition cage with a control and a treated cabbage
leaf. Each leaf (approximately 100-110 cm2) was sprayed with 0.5 mL of
MeOH or a methanolic solution of the test chemical on each side. Eggs
were counted on each cabbage leaf after 48 h. The oviposition deterrence
index (ODI) was calculated using the formula
General Information about the Spectrometers and the Conditions
1
Used To Determine H and 13C NMR Spectra. Commercial grade
solvents were distilled under nitrogen prior to use with the following
exceptions: dried THF was obtained from a MBRAUN LTS 350 solvent
purification system, and HPLC grade acetone was used without further
treatment. Reagents were used without further purification. The 1H and
13C NMR spectra were recorded in CDCl3 on Bruker 400 or 600 MHz
spectrometers. GC was run on a Hewlett-Packard 5890 using an SPB
column (Supelco, 30 m, 0.25 mm i.d., 0.25 μm film), programmed at 100 °C
(5 min) and raised at 10 °C /min to 250 °C (4.0 min). The gas chromato-
graphic data on the DB-5 column are reported as retention indices (RI).
GC mass spectra were recorded on a Varian Saturn 2000 MS coupled to a
CP 300 GC, equipped with an SPB-5 GC column (same type as above),
programmed as above. Mass spectra were acquired in EI mode [2 μscans
(0.55 s/scan), emission current (30 μamp), scanning single ion storage SIS
(m/z 49-375)]. HRMS was recorded on a 6210 series time-of-flight LC-
MS system.
Preparation of Compounds. The general procedures for the synthesis
of test compounds for the present study have been described earlier (17).
For the meta compounds, the Claisen rearrangement gave two isomers.
For the alkoxy substituents we are using, the isomer in which the allyl
group migrates to position 4 (isomer x) is slightly favored thermodyna-
mically over the isomer in which the allyl group migrates to position 2
(isomer y) (23). Typical ratios of compounds x/y range from 2.3:1 to 1.2:1.
The two isomers x and y were separated for selected cases of series 5b
(Table 4). Briefly, 1% (w/v) AgNO3 was dissolved in water and silica gel
was added to it, forming a thick slurry. The slurry dried overnight (120 °C),
before being packed into the column. Care was taken not to expose the
silver nitrate silica to light by wrapping the beaker with the slurry and later
the column with aluminum foil. The silver-silica column was equilibrated
with hexane/toluene 99:1, and the loaded compounds were eluted with
ODI ¼ ½ðC - TÞ=ðC þ TÞꢀ ꢁ 100
where C and T are the numbers of eggs laid on the control and treated leaf
disks, respectively (16).
Contact Toxicity Bioassays. Mortality was determined 24 h after
larvae had been sprayed directly with 0.25% of the test solutions (16). Third-
instar T. ni larvae were sprayed in 90 mm ꢁ 15 mm Petri dishes (Falcon)
lined with Fisher Scientific filter paper (90 mm diameter). Small plastic hand
spraying bottles (50 mL capacity) were used. Larvae were then transferred
to Petri dishes (90 mm ꢁ 15 mm) with a small piece of artificial diet. Each
Petri dish contained 10 larvae. Three replicates, each consisting of 10 larvae,
were used per treatment. Controls were sprayed with MeOH only.
Comparison of Toxicity and Oviposition and Feeding Deterrence
Values. The mortality for each test material was plotted against its
respective oviposition deterrence value (determined at 0.25%) to explore
the relationship between the two bioassays using correlation analysis.
Similarly, feeding deterrence was plotted against oviposition deterrence
and mortality (16).
Data Analysis. Feeding deterrence data (percent) from the initial
screening concentration were analyzed by analysis of variance (ANOVA)
after arcsine transformation using statistics software (24). When signifi-
cant F values were found, Tukey’s HSD multiple-comparison tests were
used to test for significant differences between individual treatments.

4986 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Akhtar et al.
Table 1. Feeding Deterrent, Oviposition Deterrent, and Toxic Bioactivities of Individual Compounds or Compound Sets to the Cabbage Looper, Based on Previous
Structure-Activity Work (16) or Precursor Sets for the Claisen Rearrangements and Subsequent Alkylations
FDb (%)
DC50c (μg/cm2)
(R2, N = 25)
mortalityd (%)
(N = 3 ꢁ 10)
ODd (%)
a
a
compound/set
R1
R2
(mean ( SE, N = 25)
(mean ( SE, N = 35-40)
3a{3,6}
3a{3,6}
3a{4,6}
3a{3,4}
3a{6,1-5}
3a{5,6}
3b{3,5}
3b{1,5}
3b{1,6}
3b{6,2-3}
3b{6,4-5}
3b{6,6}
3b{5,6}
2b{6}
allyl
allyl
allyl
propyl
propyl
butyl
96 ( 3 A
97 ( 15 A
92 ( 6 A
98 ( 2 A
68 ( 12 AB
12 (0.98)
16 (0.92)
17 (0.96)
21 (0.97)
30 (0.90)
-
17 (0.98)
29 (0.96)
34 (0.89)
42 (0.93)
32 (0.89)
20 (0.98)
-
17
10
19
19
23
-
10
30
33
65
40
13
-
28 ( 13
12 ( 13
35 ( 17
37 ( 14
67 ( 15
26 ( 13.5
9 ( 17
propyl
allyl
allyl
butyl
Me, Et, Pr, Bu, iPent
e
iPent
iPent
-
propyl
methyl
methyl
allyl
97 ( 3 A
89 ( 7 A
66 ( 12 AB
64 ( 12 AB
69 ( 11 AB
97 ( 3 A
-
iPent
allyl
30 ( 14
9 ( 14
Et, Pr
Bu, iPent
-9 ( 14
-1 ( 15
26 ( 16
10 ( 13
5 ( 13
46 ( 15
13 ( 13
17 ( 14
21 ( 14
-25 ( 12
10 ( 13
23 ( 14
allyl
allyl
allyl
iPent
allyl
H
allyl
allyl
Me, Et, Pr, Bu, iPent
36 ( 10 B
100 ( 0 A
62 ( 15 AB
73 ( 12 AB
100 ( 0 A
-
-
-
7
3c{6,1-5}
3c{1,6}
3c{2,6}
3c{3,6}
3c{5,6}
3c{2,3}
DEET
9 (0.99)
30 (0.96)
30 (0.98)
0.5 (0.98)
-
methyl
ethyl
propyl
ipent
ethyl
allyl
allyl
6
-5
-11
-
allyl
allyl
propyl
not applicable
100 ( 0 A
60 ( 15 AB
33.3 (0.99)
47 (0.98)
17
7
a Me = methyl, Et = ethyl, Pr = propyl, Bu = n-butyl, iPent = isopentyl (= 3-methylbutyl). b Feeding deterrent effects (mean ( SE) at 50 μg/cm2 are expressed in %. Means
followed by the same capital letters within a column do not differ significantly (one-way ANOVA, F18, 462 = 4.4, p < 0.0001; Tukey’s test, p < 0.05). c DC50 values (concentrations
causing 50% feeding deterrence compared with the control) were calculated for samples showing >50% feeding deterrence in initial screening concentration (50 μg/cm2), using
Excel. Linear regression analysis was conducted for all dose-response experimental data. The R2 values for the linear regressions are shown in parentheses after the number.
d Mortality and oviposition deterrent (OD) effects were determined at 0.25% for samples showing >50% feeding deterrence in initial screening. e -, not tested.
Table 2. Feeding Deterrent, Oviposition Deterrent, and Toxic Bioactivities of Libraries of Ortho Claisen Rearrangement Products from 1-Allyloxy-2-alkoxybenzenes
FDb (%)
DC50c (μg/cm2)
(R2, N = 25)
mortalityd (%)
(N = 3 ꢁ 10)
ODd (%)
a
compound/set
R2
(mean ( SE, N = 25)
(mean ( SE, N = 35-40)
4a{1-5}
H
100 ( 0 A
100 ( 0 A
100 ( 0 A
31 ( 15 B
6 ( 17 B
20.6 (0.92)
15.5 (0.86)
20.0 (0.91)
17
17
27
47
1
7 ( 15
32 ( 15
31 ( 15
-
-
-
5a{1,1-5}
5a{2,1-5}
5a{3,1-5}
5a{4,1-5}
5a{5,1-5}
5a{6,1-5}
Me
Et
e
Pr
Bu
-
-
-
-
iPent
allyl
22 ( 16 B
22 ( 18 B
7
7
-
a Me = methyl, Et = ethyl, Pr = propyl, Bu = n-butyl, iPent = isopentyl (= 3-methylbutyl). For 5a sets, the code is 5a{R2,R1} (Figure 1). b Feeding deterrent effects (mean ( SE)
at 50 μg/cm2 are expressed in %. Means followed by the same capital letters within a column do not differ significantly (one-way ANOVA, F6, 173 = 12.5, p < 0.0001; Tukey’s test,
p < 0.05). c DC50 values (concentrations causing 50% feeding deterrence compared with the control) were calculated for samples showing >50% feeding deterrence in initial
screening concentration (50 μg/cm2), using Excel. Linear regression analysis was conducted for all dose-response experimental data. The R2 values for the linear regressions
are shown in parentheses after the number. d Mortality and oviposition deterrent (OD) effects were determined at 0.25% for samples showing >50% feeding deterrence in initial
screening. e -, not tested.
RESULTS
At 0.25% the 1-allyloxy-3-ethoxy/-propoxybenzene set 3b-
{6,2-3} was the most toxic (65% mortality,) followed by the
1-allyloxy-3-butoxy/isopentoxybenzene set 3b{6,4-5} (40%
mortality) (Table 1). 1-Allyloxy-4-ethoxybenzene 3c{2,6} was
the least toxic (6.7% mortality) in this group.
At 0.25% the 1-allyloxy-2-alkoxybenzene set 3a{6,1-5} showed
the strongest oviposition deterrent effect (66.7%) (Table 1). Inter-
estingly, members of set 3c{6,1-5} with small alkoxy substituents
(3c{1,6}, 3c{2,6}, and 3c{3,6}) were poor oviposition deterrents
Feeding Deterrent, Toxicity, and Oviposition Deterrent Effects.
Individual Compounds or Compound Sets, Based on Previous
Structure-Activity Work (16). DC50 values varied from 0.5 to
42.1 μg/cm2 (Table 1). The compound 1-allyloxy-4-propoxyben-
zene, 3c{3,6}, had the lowest DC50 value (0.5 μg/cm2) followed by
the 1-allyloxy-4-alkoxybenzene set 3c{6,1-5}, (8.5 μg/cm2),
which contains compound 3c{3,6}. DEET showed a DC50 value
of 46.7 μg/cm2 (Table 1).

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J. Agric. Food Chem., Vol. 58, No. 8, 2010 4987
Table 3. Feeding Deterrent, Oviposition Deterrent, and Toxic Bioactivities of Libraries of Ortho Claisen Rearrangement Products from 1-Allyloxy-4-alkoxybenzenes
FDb (%)
DC50c (μg/cm2)
(R2, N = 25)
mortalityd (%)
(N = 3 ꢁ 10)
ODd (%)
a
compound/set
R2
(mean ( SE, N = 25)
(mean ( SE, N = 35-40)
4c{1-5}
5c{1,1}
H
Me, R1 = Me
51 ( 18 ABC
80 ( 11 AB
68 ( 12 ABC
66 ( 14 ABC
92 ( 6 A
57 (0.92)
20 (0.88)
19 (0.94)
22 (0.95)
9.4 (0.90)
33 (0.98)
3.3
39
51 ( 17
14 ( 12
37 ( 15
64 ( 17
7 ( 15
21 ( 14
-14 ( 13
20 ( 15
-
5c{1,1-5}
5c{2,1-5}
5c{3,1}
Me
Et
17
3.3
6.4
17
Pr, R1 = Me
Pr
Bu
5c{3,1-5}
5c{4,1-5}
5c{5,1-5}
5c{6,1-5}
6c{1-5}
65 ( 11 ABC
23 ( 16 C
e
-
15 (0.85)
-
27
iPent
allyl
83 ( 12 AB
29 ( 11 BC
100 ( 0 A
-
16 (0.95)
-
1.0
cyclic
-12 ( 13
a Me = methyl, Et = ethyl, Pr = propyl, Bu = n-butyl, iPent = isopentyl (= 3-methylbutyl). For 5c sets, the code is 5c{R2,R1} (Figure 1). b Feeding deterrent effects (mean ( SE)
at 50 μg/cm2 are expressed in %. Means followed by the same capital letters within a column do not differ significantly (one-way ANOVA, F9-241 = 4.3, p < 0.0001; Tukey’s test, p <
0.05). c DC50 values (concentrations causing 50% feeding deterrence compared with the control) were calculated for samples showing >50% feeding deterrence in initial
screening concentration (50 μg/cm2), using Excel. Linear regression analysis was conducted for all dose-response experimental data. The R2 values for the linear regressions
are shown in parentheses after the number. d Mortality and oviposition deterrent (OD) effects were determined at 0.25% for samples showing >50% feeding deterrence in initial
screening. e -, not tested.
(<30%). The meta-substituted dialkoxybenzenes (3b compounds)
isomeric compounds (Table 4), exhibited the lowest DC50 value
(4 μg/cm2), in one trial. A different lot of set 5b{5,1} exhibited a
higher DC50 value (16 μg/cm2). With respect to feeding deterrence
there were clear structure-activity relationships. For group R2,
propyl gave the best results, and the smaller (methyl, ethyl, or
allyl) or larger (butyl or isopentyl) groups gave lower feeding
deterrence. For group R1 the structure-activity relationship was
clear: within each group with R2 constant, there was a decrease in
activity in going from R1 = methyl to the larger groups. For cases
in which isomers x and y were separated, the more compact
isomer y was more active as a feeding deterrent than isomer x.
Compound 4b{2-3} was the most toxic, causing 70% mortal-
ity at 0.25% (Table 4). Thus, group R2 = H or methyl gave high
mortality. For the larger R2 sets, mortality was lower, and there
was a slight pattern with respect to group R1 within each group
with constant R2 (ethyl or propyl): the set with R1 = ethyl/propyl
was more toxic than the set with R1 = methyl.
5b{5,1} and 5b{6,1} demonstrated the strongest oviposition
deterrent effects (50%). Set 5b{2,4-5} acted as a mild oviposition
stimulant (Table 4). There were clear structure-activity patterns
in the oviposition data. Among the 4b sets, oviposition deterrence
increased with increasing size of group R1. Also in the 5b sets
when R2 = methyl, ethyl, or propyl, there was an increase in
oviposition deterrence going from R1 = methyl to R1 = ethyl/
propyl, but when R2 = isopentyl or allyl, there was a decrease in
oviposition deterrence going from R1 = methyl to R1 = ethyl/
propyl or butyl/isopentyl. In the R2 = butyl sets, oviposition
deterrence was the same for all R1 groups.
were generally weak, with the strongest congeners being the ones
3
˚
with a molecular volume of 250-260 A and either an allyloxy or
an isopentyloxy group 3b{1,5} and 3b{6,6} (25-30% oviposition
deterrence).
Libraries of Ortho Claisen Rearrangement Products from
1-Allyloxy-2-alkoxybenzenes. The 5a{1,1-5} minilibrary had
the lowest DC50 value (16 μg/cm2) followed by 5a{2,1-5} and
4a{1-5} (∼20 μg/cm2) (Table 2). A structure-activity relation-
ship was clear among these compounds: small R2 groups (H,
methyl, or maximally ethyl) gave high feeding deterrence. This
activity was lost significantly with a one or more carbon increase
in the size of group R2.
Set 5a{3,1-5} was the most toxic (Table 2) at 0.25% (47%
mortality). A structure-activity relationship could be seen, with
R2 = propyl being most toxic and R2 groups smaller or larger
than that being less toxic. At 0.25% the sets 5a{1,1-5}and
5a{2,1-5} caused ∼30% oviposition deterrence (Table 2).
Libraries of Ortho Claisen Rearrangement Products from
1-Allyloxy-4-alkoxybenzenes. Set 5c{3,1} had the lowest DC50
value (9 μg/cm2), whereas 4c{1-5} had the highest DC50 value
(57 μg/cm2). There was a moderate structure-activity relation-
ship among the sets 5c{R2,1-5}, with R2 = butyl or allyl being
less active than R2 = methyl, ethyl, propyl, or isopentyl.
Compounds 5c{3,1} and 5c{1,1} were more active than the entire
5c{3,1-5} or 5c{1,1-5} sets, respectively.
At 0.25% set 5c{1,1} was the most toxic with 38.9% mortality
followed by 5c{5,1-5} (Table 3). Other members in the group
exhibited <26% mortality (Table 3). Set 5c{2,1-5} demon-
strated strong oviposition deterrent activity (63.6%) followed
by 4c{1-5} (51.4%) and 5c{1,1-5} (37%). There was some
structure-activity relationship with respect to oviposition deter-
rence, with the optimal R2 alkyl group being ethyl: smaller or
larger was less effective. Set 6c{1-5} acted as a moderate
oviposition stimulant. All other libraries had only modest ovipo-
sition deterrent activities (Table 3).
Eugenol and Alkylated Derivatives of Eugenol. Butyl and
eugenol exhibited the lowest DC50 value (12 μg/cm2) followed by
allyleugenol (15 μg/cm2). There was an increase in feeding
deterrence (and a decrease in DC50), with increasing size of group
R1 from methyl to butyl. The larger isopentyl group gave slightly
lower feeding deterrence (higher DC50) (Table 5).
Methyleugenol was the most toxic, causing 41% mortality at
0.25% (Table 5). All others exhibited <37% mortality. There
was a structure-activity relationship for toxicity as well, with an
increase from R1 = H (eugenol) to methyleugenol, but then a
Libraries of Ortho Claisen Rearrangement Products from
1-Allyloxy-3-alkoxybenzenes. Set 5b{5,1}, a mixture of two

4988 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Akhtar et al.
Table 4. Feeding Deterrent, Oviposition Deterrent, and Toxic Bioactivities of Libraries of Ortho Claisen Rearrangement Products from 1-Allyloxy-3-alkoxybenzenes
FDb (%)
DC50c (μg/cm2)
(R2, N = 25)
mortalityd (%)
(N = 3 ꢁ 10)
ODd (%)
compound/seta
4b{1}
R1
Me
R2
(mean ( SE, N = 25)
(mean ( SE, N = 35-40)
a
a
H
74 ( 11 ABC
73 ( 11 ABC
66 ( 13 ABC
77 ( 9 ABC
74 ( 11 ABC
41 ( 16 ABC
93 ( 5 AB
25 (0.98)
33 (0.99)
26 (0.96)
25 (0.99)
23 (0.98)
20
70
50
50
65
-
3.6
20
10
3.6
10
-
0.6
10
6.7
-
16
6.7
-
12 ( 12
31 ( 14
45 ( 15
-5 ( 13
19 ( 15
-
-2 ( 13
28 ( 14
-24 ( 13
-11 ( 15
5 ( 13
34 ( 15
25 ( 13
13 ( 14
9 ( 13
18 ( 14
51 ( 14
15 ( 14
-
4b{2-3}
4b{4-5}
5b{1,1}
Et, Pr
Bu, iPent
Me
H
H
Me
Me
Me
Et
5b{1,2-3}
5b{1,4-5}
5b{2,1}
Et, Pr
Bu, iPent
Me
h
-
24 (0.89)
17 (0.99)
15 (0.95)
16 (0.90)
8.7 (0.85)
-
17 (0.79)
27 (0.90)
49 (0.96)
-
19 (0.93)
24 (0.98)
-
21 (0.97)
16 (0.96)
43 (0.96)
5b{2,2-3}
5b{2,4-5}
5b{3,1}
Et, Pr
Bu, iPent
Me
Et
79 ( 10 ABC
81 ( 11 ABC
100 ( 0 A
Et
Pr
Pr
Pr
Pr
Bu
Bu
Bu
5b{3,2-3}
5b{3,4-5}
5b{3,2}
Et, Pr
Bu, iPent
Et
93 ( 5 AB
33 ( 16 BC
92 ( 6 AB
5b{4,1}
Me
60 ( 16 ABC
54 ( 16 ABC
26 ( 16 C
5b{4,2-3}
5b{4,4-5}
5b{5,1}
Et, Pr
Bu, iPent
Me
iPent
iPent
iPent
allyl
71 ( 14 ABC
79 ( 9 ABC
41 ( 17 ABC
76 ( 12 ABC
90 ( 8 AB
5b{5,2-3}
5b{5,4-5}
5b{6,1}
Et, Pr
Bu, iPent
Me
25
33
33
50 ( 14
8 ( 13
19 ( 13
5b{6,2-3}
5b{6,4-5}
Et, Pr
Bu, iPent
allyl
allyl
57 ( 15 ABC
second lots and purified isomers of 5b{n,1} or 5b{3,2} compounds
5b{1,1}e
Me
Me
Me
Me
Et
Me
Pr
58 ( 15 ABC
91 ( 6 AB
100 ( 0 A
68 ( 12 ABC
92 ( 6 AB
74 ( 14 ABC
95 ( 3 AB
99 ( 1 A
39 (0.97)
26 (0.96)
14 (0.98)
24 (0.76)
17 (0.79)
28 (0.99)
25 (0.98)
4 (0.98)
-2.9
10
10 ( 15
7 ( 11
17 ( 14
0
5b{3,1}e
5b{3,1}y (100% y)
5b{3,1}x (68% x, 32% y)
5b{3,2}g
Pr
-4.2
-32
0.6
Pr
Pr
25 ( 13
-6.4 ( 11.4
26 ( 8
32 ( 12
10 ( 14
5b{3,2}y (100% y)
5b{3,2}x (82% x, 18% y)
5b{5,1}e
5b{6,1}e
Et
Pr
0
Et
Pr
0
Me
Me
iPent
allyl
6.0 (
-1.6 (
92 ( 6 AB
23 (0.92)
a Me = methyl, Et = ethyl, Pr = propyl, Bu = n-butyl, iPent = isopentyl (= 3-methylbutyl). For 5b sets, the code is 5b{R2,R1} (Figure 1) . The compounds are a mixture of isomers
x and y in a ratio of x/y 2:1. b Feeding deterrent effects (mean ( SE) at 50 μg/cm2 are expressed in %. Means followed by the same capital letters within a column do not differ
significantly (one-way ANOVA, F27, 684 = 3.2, p < 0.0001; Tukey’s test, p < 0.05). c DC50 values (concentrations causing 50% feeding deterrence compared with the control) were
calculated for samples showing >50% feeding deterrence in initial screening concentration (50 μg/cm2), using Excel. Linear regression analysis was conducted for all
dose-response experimental data. The R2 values for the linear regressions are shown in parentheses after the number. d Mortality and oviposition deterrent (OD) effects were
determined at 0.25% for samples showing >50% feeding deterrence in initial screening. e These lots were prepared on a larger scale than previously (17), and the ratios of x/y were
as follows: 5b{1,1}, 1.8:1; 5b{3,1}, 1.2:1; 5b{3,2}, 2.3:1; 5b{5,1} 1.8:1; 5b{6,1}, 2.3:1. f The isomers were separated on a column of silica/silver nitrate (see Materials and
Methods). g Same set as listed above with the sets, provided for convenience. h -, not tested.
general decrease with increasing group size from methyl to butyl.
The larger isopentyleugenol was again slightly more toxic.
Butyl and eugenol demonstrated the strongest oviposition
deterrent effect (34%) followed by propyl and eugenol (29%)
when tested at 0.25%. Methyl and eugenol acted asan oviposition
stimulant (Table 5). There was a structure-activity pattern here,
too: as the alkyl group increased, oviposition deterrence generally
increased, up to butyleugenol. Isopentyleugenol and allyleugenol
were less active in this category.
Comparison of Toxicity and Oviposition and Feeding Deterrence.
There was no correlation between toxicity and oviposition deter-
rence (y = -0.0738x þ 20.582, R2 = 0.0044) within the data sets.
Similarly, there was no correlation between feeding deterrence
and oviposition deterrence (y = -0.2005x þ 25.333, R2 = 0.0368)
within the data sets. Furthermore, there was no correlation
between toxicity and feeding deterrence (y = -0.0235x þ 20.565,
R2 = 0.0006).
Comparison of Feeding Deterrence with Long-Term EAG In-
hibition of Sex Attractant Pheromone Responses in Male Gypsy
Moth (Another Lepidopteran Species). To our surprise, we found a
correlation between larval feeding deterrence in third-instar T. ni
larvae and long-term EAG inhibition of sex attractant phero-
mone responses in male gypsy moth, Lymantria dispar (y = 2.0x -
2.9, R2= 0.66, Figure 2) (25) for some of the compounds.
Compounds that were active in both insects are (in order of
decreasing activity) 3c{2,3}, 3c{3,1-5}, 3c{4,1-5}, 3a{4,4},
3b{3,3}, 3c{5,1-5}, 3c{3,6}, 2a{4}, 3b{1,1-5}, 5a{3,1-5},
2b{5}, 2c{1}, 2c{3}, 2a{1}, 3c{3,3}, and 2b{2}.
DISCUSSION
Our results have demonstrated that most of the compounds
and libraries are effective feeding deterrents, with minimal toxicity
in most cases. We are focusing on compounds that are behaviorally
active without acute toxicity to the insects as we anticipate that

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 4989
Table 5. Feeding Deterrent, Oviposition Deterrent, and Toxic Bioactivities of Eugenol and Alkylated Derivatives of Eugenol
a
compound
eugenol
R1
FDb (%) (mean ( SE, N = 25)
DC50c (μg/cm2) (R2, N = 25)
mortalityd (%) (N = 3 ꢁ 10)
ODd (%) (N = 35-40)
e
-
H
44 ( 16 A
64 ( 15 A
51 ( 18 A
84 ( 11 A
86 ( 9 A
56 (0.89)
26 (0.90)
50 (0.88)
19 (0.92)
15 (0.94)
12 (0.89)
25 (0.99)
30
41
35
37
18
15
28
methyleugenol
ethyleugenol
Me
Et
-22 ( 14
18 ( 12
29 ( 15
16 ( 16
34 ( 14
9 ( 14
propyleugenol
allyleugenol
Pr
allyl
Bu
n-butyleugenol
isopentyleugenol
92 ( 8 A
iPent
86 ( 10 A
a Me = methyl, Et = ethyl, Pr = propyl, Bu = n-butyl, iPent = isopentyl (= 3-methylbutyl). b Feeding deterrent effects (mean ( SE) at 50 μg/cm2 are expressed in %. Means
followed by the same capital letters within a column do not differ significantly (one-way ANOVA, F6,171 = 2.07, p > 0.06; Tukey’s test, p < 0.05). c DC50 values (concentrations
causing 50% feeding deterrence compared with the control) were calculated for samples showing >50% feeding deterrence in initial screening concentration (50 μg/cm2), using
Excel. Linear regression analysis was conducted for all dose-response experimental data. The R2 values for the linear regressions are shown in parentheses after the number.
d Mortality and oviposition deterrent (OD) effects were determined at 0.25% for samples showing >50% feeding deterrence in initial screening. e -, not tested.
Figure 2. Correlation of the activities of compounds from this and a
previous (16) study: feeding deterrence (%) against T. ni larvae versus
long-term inhibition of EAG responses to the sex pheromone in L. dispar
males (25). Compounds for which the two activities paralleled are shown in
pink, and the line shown refers to these compounds.
Figure 3. (A) “Site” defined by structure-activity relationships for feeding
deterrence of T. ni third-instar larvae (this study and ref 16) and on the
inhibition of the EAG responses to the sex pheromone of L. dispar males
(25). (B) Set of overlapping “sites” defined by structure-activity relation-
ships for oviposition deterrence of T. ni adult females.
these will have low impact on nontarget arthropods and fewer
regulatory obstacles. Most of them possess low or medium
oviposition deterrent effects, and a few act as oviposition stimu-
lants. The highest feeding deterrence was seen for compound
3c{3,6}. It was considered to be the best candidate for manipulat-
ing larval feeding, as it exhibited low toxicity and oviposition
deterrence to the cabbage looper larvae and female moths,
respectively. Compounds 5c{3,1} and 5c{1,1} were more active
than the entire 5c{3,1-5} or 5c{1,1-5} sets, respectively. Because
sets and compounds were tested at the same concentration by
weight, this result suggests that the activity detected for the sets
came mostly from the most active component, and there is
probably little or no synergy or antagonism within these sets.
Overall, the structure-activity suggests that good feeding deter-
rents in the 5c group have an odd-numbered (methyl or propyl) or
branched (isopentyl) R2 alkyl group and a small R1 (methyl)
group. Compound set 6c{1-5} was formed during the synthesis
of 5c libraries (in cases when the Claisen reaction was left too
long) (17). The cyclic portion of compounds 6c{1-5} resembles a
branched chain and could fit the same type of site as the 5c
compounds.
and 5b{5,1} also had high oviposition deterrence activity, and of
these 4c{1-5} had the lowest toxicity. All of these oviposition
deterrents were moderate or poor feeding deterrents, so these
candidates would target only the oviposition behavior. Finally,
set 5a{1,1-5} and the compound butyleugenol were not the best
overall in any of the activities tested, but they best combined all
three desirable properties in one: high feeding deterrence, high
oviposition deterrence, and low toxicity.
One important aspect of our study is that many of the pure
compounds and libraries were more active than a commercial
insect repellent, DEET, as feeding and/or oviposition deterrents
against T. ni larvae and adult female moths, respectively. DEET
exhibited the highest DC50 value (47 μg/cm2) in the whole group
(Table 1) as opposed to the low DC50 value of 0.5 μg/cm2
exhibited by 3c{3,6}. DEET is a highly effective (26) and widely
used insect repellent. We used DEET as a positive control in our
study. The advantage of having compared our results to DEET is
that one target site for DEET is known in the dipterans: DEET
interacts with the conserved olfactory coreceptor OR83b (27).
The lepidopteran orthologue of this protein has been designated
OR2 (28). Interaction of DEET with OR83b and other olfactory
The highest oviposition deterrence was seen with set 3a-
{6,1-5}, and this set may exhibit synergistic effects, because
compound 3a{5,6} in isolation showed some oviposition deter-
rence, but lower than that of the full set. Sets 4c{1-5}, 4b{4-5},

4990 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Akhtar et al.
components, such as the specific OR for a particular set of
odors (27, 28), causes the inhibition of behavioral attraction to
food odors in Drosophila melanogaster and Anopheles gam-
biae (29).
effect. The nontoxic deterrents found here could be used in
combined strategies, in which the larvae or the females are
deterred from the crop and attracted to traps or refuge areas, as
shown in a previous study (32).
Because our leading candidate compound for feeding deter-
rence, 3c{3,6}, has some resemblance to DEET and appears to be
highly active on more than one species and more than one life
stage of Lepidoptera (see above), there is a possibility that this
compound also acts on one of the widespread, conserved com-
ponents of the olfactory system.
The most active feeding deterrents overlap on a site that could
also accommodate DEET (Figure 3A). Such a site could be
located on a single protein or at a protein/protein interface needed
for the olfactory response. Interactions between various dendritic
membrane components have been shown to be important for
olfactory responses in insects. For example, it has been shown
with Drosophila that the coreceptor OR83b interacts very closely
with the sensory neuron membrane protein (SNMP) (30) and
with the odorant receptor (OR) itself (29).
Compounds and sets 3a{3,1-5}, 3a{4,1-5}, 3b{4,1-5}, 3b-
{5,1-5}, 3c{6,6}, 3c{1,1-5}, 3c{2,1-5}, 4b{1}, 5a{1,1-5}, 5a-
{2,1-5}, 5b{2,4-5}, 5b{3,2-3}, 5b{6,2-3}, 5b{3,1}, 5b{6,1},
5c{5,1-5}, allyleugenol, propyleugenol, and butyleugenol
showed strong feeding deterrence activity in T. ni larvae in the
present study, but were not active as inhibitors of pheromone
perception in L. dispar adult males (25). These compounds or sets
may be perceived more specifically as “unpleasant” general
odorants or tastants by T. ni larvae. Finally, the oviposition
deterrents seem to target a set of overlapping sites (Figure 3B) that
cause deterrence if occupied by the alkyl and allyl groups on the
same side of the benzene ring (ortho positions) and oviposition
stimulation if occupied by the alkyl and allyl groups on opposite
sides of the benzene ring (meta and para positions).
On the basis of antifeedant activity, our compounds/libraries
possess levels of activity that compare favorably to some of the
most active botanical insecticides in current use. Compound
3c{3,6} in the group is as active as pyrethrum (DC50 = 0.9 μg/
cm2) in the feeding deterrence bioassay with T. ni larvae (31).
Similarly, other compounds/sets including 5c{3,1}, 3c{6,1-5},
5b{3,2-3}, and 5b{5,1}x þ y were more active than rotenone
against third-instar T. ni larvae. All of the candidate compounds/
sets were more active than rosemary oil (DC50 = 158 μg/cm2),
clove leaf oil (DC50 = 217 μg/cm2), Melia azedarach (DC50 = 288
μg/cm2), Trichilia americana (DC50 = 190 μg/cm2), and Ryania
(DC50 = 725 μg/cm2) (31).
In conclusion, we have described the activity of synthetic
aromatic compounds that mediate the feeding and oviposition
behavior of cabbage looper larvae and adult female moths,
respectively. On the basis of their comparable efficacy to azadir-
achtin and other commercial feeding deterrents, many of the
compounds/libraries have potential for development as commer-
cial insect control agents with selectivity toward Lepidoptera.
However, the demonstration of bioactivity in the laboratory is
simply the first step in the development of a commercial product,
and numerous other criteria must be satisfied before the true
commercial potential can be realized (33). Empirical tests are
needed to confirm low nontarget toxicity (especially low mam-
malian toxicity), and persistence under field conditions needs to
be assessed. Persistence and other aspects of field performance
can be partly addressed through proper formulation, provided
that solvents and adjuvants used are compatible with conven-
tional application equipment and can maintain a cost to the end-
user that is competitive with that of other pest management
products. The failure to meet one or more of these criteria
accounts for the dearth of alternative insect control products
(i.e., those not based on neurotoxins). The next step in our
research will be focused on determining the efficacy of the
compounds in a greenhouse environment.
ACKNOWLEDGMENT
We thank Nancy Brard for insect rearing and technical
assistance and Jocelyn Greer for assistance with bioassays. We
thank Chloe Coppin and Andrew Round for assistance with the
synthesis of pure candidate compounds.
Supporting Information Available: Data for the individual
compounds and 1H NMR data of new, pure 3a, 3b, and 3c
compounds that have not been published previously (17). This
material is available free of charge via the Internet at http://pubs.
acs.org.
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(21) Wang, P.; Zhao, J. Z.; Rodrigo-Simon, A.; Kain, W.; Janmaat, A. F.;
Received for review December 21, 2009. Revised manuscript received
February 24, 2010. Accepted February 26, 2010. This research was
supported by NSERC Strategic Grant STPGP 307515 (to E.P. and
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